Marburg virus (MBGV) was first recognized in 1967, when an outbreak of hemorrhagic fever in humans occurred in Germany and Yugoslavia, after the importation of infected monkeys from Uganda (Martini and Siegert, 1971, Marbura Virus Disease. Berlin: Springer-Verlag; Smith et al., 1982, Lancet 1, 816-820). Thirty-one cases of MBGV hemorrhagic fever were identified that resulted in seven deaths. The filamentous morphology of the virus was later recognized to be characteristic, not only of additional MBGV isolates, but also of Ebola virus (EBOV) (Johnson et al., 1977, J. Virol. 71, 3031-3038; Smith et al., 1982, Lancet 1, 816-820; Pattyn et al., 1977, Lancet 1, 573-574). MBGV and EBOV are now known to be distinctly different lineages in the family Filoviridae, within the viral order Mononegavirales (Kiley et al., 1982, Intervirology 18, 24-32; Feldmann and Klenk, 1996, Adv. Virus Res. 47, 1-52).
Few natural outbreaks of MBGV disease have been recognized, and all proved self-limiting, with no more than two cycles of human-to-human transmission. However, the actual risks posed by MBGV to global health cannot be assessed because factors which restrict the virus to its unidentified ecological niche in eastern Africa, and those that limit its transmissibility, remain unknown (Feldmann and Klenk, 1996, supra). Concern about MBGV is further heightened by its known stability and infectivity in aerosol form (Belanov et al., 1996, Vopr. Virusol. 41, 32-34; Frolov and Gusev Iu, 1996, Vopr. Virusol. 41, 275-277). Thus, laboratory research on MBGV is necessarily performed at the highest level of biocontainment. To minimize future risk, our primary interest has been the identification of appropriate antigens and vaccine strategies that can provide immunity to MBGV.
Early efforts to demonstrate the feasibility of vaccination against MBGV were only partially successful, as inoculation with formalin-inactivated viruses only protected about half the experimental animals (guinea pigs or nonhuman primates) from fatal disease (Ignat""ev et al., 1991, Vopr. Virusol. 36, 421-423; Ignat""ev et al., 1996, J. Biotechnol. 44, 111-118). We recently demonstrated that the MBGV GP, cloned into a baculovirus vector and expressed as a soluble antigen to be administered in adjuvant, was sufficient to protect most but not all guinea pigs from lethal MBGV challenge (Hevey et al., 1997, Virology 239, 206-216). In addition, purified, 60Co-irradiated virus, administered in adjuvant, completely protected guinea pigs from challenge with either of two different strains of MBGV, thus setting a standard for future, more pragmatic, vaccine candidates (Hevey et al., 1997, supra). Experiences with EBOV vaccines have been similar to those with MBGV, reinforcing the difficulties of classical approaches (Lupton et al., 1980, Lancet 2, 1294-1295). Recent efforts to develop EBOV vaccines, using three distinctly different approaches (vaccinia recombinants, VEE replicon, and naked DNA) to achieve viral antigen expression in cells of vaccinated animals, showed that nucleoprotein (NP) as well as GP protected BALB/c mice (VanderZanden et al., 1998, Virology 245), whereas protection of guinea pigs by NP was unsuccessful (Gilligan et al., 1997, In: Brown, F., Burton, D., Doherty, P., Mekalanos, J., Norrby, E. (eds). 1997. Vaccines 97 Cold Spring Harbor Press. Cold Spring Harbor, N.Y.; Pushko et al., 1997, In: Brown, F., Burton, D., Doherty, P., Mekalanos, J., Norrby, E. (eds). 1997. Vaccines 97 Cold Spring Harbor Press. Cold Spring Harbor, N.Y.) or equivocal (Xu et al., 1998, Nat. Med. 4, 37-42).
Irrespective of how encouraging filovirus vaccine results may appear in guinea pigs or mice, protection of nonhuman primates is widely taken as the more definitive test of vaccine potential for humans. Low-passage viral isolates from fatal human cases of MBGV or EBOV tend to have uniform lethality in nonhuman primates, but not in guinea pigs or mice. Small animal models with fatal disease outcomes have been achieved only with a subset of filovirus isolates and only then by multiple serial passages in the desired host (Hevey et al., 1997, supra; Connolly et al., 1999, J. Infect. Dis. 179, suppl. 1, S203; Xu et al., 1998, supra; Bray et al., 1998, J. Infect. Dis. 178, 661-665). While highly useful for identification and initial characterization of vaccine candidates, guinea pig and murine models remain somewhat suspect with regard to the possibility that protection in such animals is easier to achieve than in nonhuman primates and, by inference, in humans. For example, with MBGV, peak viremias and viral titers in organs are more than 100 times higher in nonhuman primates than in guinea pigs.
Therefore, there is a need for an efficacious vaccine for MBGV useful for protecting humans against Marburg hemorrhagic fever.
The present invention satisfies the need discussed above. The present invention relates to a method and composition for use in inducing an immune response which is protective against infection with MBGV.
In this study a vaccine delivery system based on a Venezuelan equine encephalitis (VEE) virus replicon was used to identify candidate protective antigens in nonhuman primates. In this vaccine strategy, a gene coding for a protein of interest is cloned in place of the VEE virus structural genes; the result is a self-replicating RNA molecule that encodes its own replicase and transcriptase functions, and in addition makes abundant quantities of mRNA encoding the foreign protein. When replicon RNA is transfected into eukaryotic cells along with two helper RNAs that express the VEE structural proteins (glycoproteins and nucleocapsid), the replicon RNA is packaged into VEE virus-like particles by the VEE virus structural proteins, which are provided in trans. Since the helper RNAs lack packaging signals neccessary for further propagation, the resulting VEE replicon particles (VRPs) which are produced are infectious for one cycle but are defective thereafter. Upon infection of an individual cell with a VRP, an abortive infection occurs in which the infected cell produces the protein of interest in abundance, is ultimately killed by the infection, but does not produce any viral progeny (Pushko et al., 1997, Virology 239, 389-401). The VEE replicon is described in greater detail in U.S. Pat. No. 5,792,462 issued to Johnston et al. on Aug. 11, 1998.
Results shown here demonstrate that the VEE replicon is a potent tool for vaccination with MBGV antigens. Guinea pigs were protected by vaccination with packaged replicons that expressed GP, or by either of two replicons which expressed internal MBGV antigens (NP and VP35). GP expressed from the VEE replicon elicited an even more robust immunity than was achieved previously with a baculovirus-produced soluble GP administered in adjuvant. When results were extended to nonhuman primates, complete protection with GP was demonstrated. The data shown here constitute the most emphatic proof to date that an efficacious vaccine for MBGV is feasible, and define candidate antigens for such a vaccine.
Therefore, it is one object of the present invention to provide a VEE virus replicon vector comprising a VEE virus replicon and a DNA fragment encoding any of the MBGV GP, NP, VP40, VP35, VP30, and VP24, and GPxcex94TM, a GP deletion mutant from which the C-terminal 39 amino acids encoding the transmembrane region and cytoplasmic tail of MBGV GP were removed.
It is another object of the present invention to provide a self replicating RNA comprising the VEE virus replicon and any of the MBGV GP, GPxcex94TM, NP, VP40, VP35, VP30, and VP24 described above.
It is another object of the present invention to provide infectious VEE virus replicon particles produced from the VEE virus replicon RNA described above.
It is further an object of the invention to provide an immunological composition for the protection of mammals against MBGV infection comprising VEE virus replicon particles containing nucleic acids encoding any of the MBGV GP, GPxcex94TM, NP, VP40, VP35, VP30, and VP24 or a combination of different VEE virus replicons each containing nucleic acids encoding a different MBGV protein from any of MBGV GP, GPxcex94TM, NP, VP40, VP35, VP30, and VP24.